Human African trypanosomiasis (HAT) is a neglected tropical disease (NTD) that is caused by the protozoan parasite Trypanosoma brucei, and for which current drugs have poor efficacy or toxicity profiles. Discovery of new drugs for this fatal disease has been hindered by a lack of high quality lead compounds for optimization. In a unique industrial-academic collaboration, the Principal Investigators have been collaborating with GlaxoSmithKline (GSK) in a project funded by the Tres Cantos Open Lab Foundation, which has enabled the high-throughput screen (HTS) of over 46,000 kinase-targeted inhibitor compounds against T brucei cells. The project resulted in discovery of 797 potent (T brucei EC50< 1 ?M) and selective (> 100-fold over HepG2 cells) that were sorted into 59 structural clusters, plus 53 singleton compounds. With a desire to prioritize this large set of hit compounds, these compounds were assessed for their rate of action, reversibility of growth inhibition, and for a wide range of physicochemical and drug metabolism properties (computed and measured). This proposal outlines a hit-to-lead medicinal chemistry program that will optimize four of the highest- priority hit clusters, plus a highly potent singleton compound that shows efficacy in T brucei bloodstream infections in mice. Optimization will result in the delivery of high-quality lead compounds from each of these five chemical classes, and these leads will meet stringent profiles of cellular potency and selectivity, blood- brain barrier permeability, physicochemical and metabolic properties, and pharmacokinetic properties in mice. Central nervous system (CNS) exposure is of paramount importance. Furthermore, these lead series will display in vivo efficacy in murine models of bloodstream and CNS T brucei infections. The optimization program described in this proposal will be performed under the continuing collaboration between the PIs at Northeastern University and the Spanish National Council for Research (CSIC), with critical contributions of expertise and drug metabolism and physicochemical properties experiments from GSK. We have developed and implemented a testing funnel that ensures that the optimization process will address the most critical lead criteria. With an interest in seeding others' work in HAT drug discovery, all data generated in the course of this program will be disclosed in real time via a searchable database that can be accessed by NTD researchers world-wide. Taken together, we believe that this represents an unprecedented resource for HAT drug discovery. In the end, this project will deliver (a) multiple lead compounds for HAT that meet well-defined Lead Criteria; (b) broader profiling of lead compounds against more stringent Candidate Criteria (enabling evaluation by the Drugs for Neglected Disease Initiative (DNDi) for potential further advancement. In that way, we intend to help fill the early preclinical drug discovery pipeline for HAT.